Use of light scattering particles in design, manufacture, and quality control of small volume instruments, devices, and processes

ABSTRACT

The use of light scattering particles in the design, manufacturing, and quality control of microscale devices and process, and the analysis of solid substrate and porous substrate characteristics is described.

BACKGROUND OF THE INVENTION

[0001] The present invention concerns the field of development andquality control of devices and materials for fluid handling, control,flow, and deposition.

[0002] At presen methods for analyses in these applications typicallyrely upon detection of fluorescent or chromogenic dyes using atime-lapse or high-speed video device. For flow analysis, a fine streamof the fluorescent or chromogenic detection agent can be introduced intothe system. However, information obtained using these methods is oflimited utility with respect to a significant area of the flow channelbecause of limited sensitivity and rapid diffusion and dilution of thedetected agent.

[0003] Resonance light scattering (RLS) particles have been shown toprovide highly sensitive labels in bioanalytical assays in a variety ofdifferent formats. Such uses are described, for example, in Yguerabideat al., PCT/US97/06584, Yguerabide et al., PCT/US98/23160, Yguerabide etal, U.S. application Ser. No. 08/844,217, now U.S. Pat. No. 6,214,560,and Yguerabide et al., U.S. application Ser. No. 08/953,713, and thepatents and publications cited in the backgrounds thereof, all of whichare hereby incorporated by reference herein in their entireties,including drawings.

[0004] Particles with similar composition have also been used inconnection with electron microscopy and as cytological stains, utilizingtheir associated absorbence properties.

SUMMARY OF THE INVENTION

[0005] The present invention relates to the use of resonance lightscattering (RLS) particles as tools to guide and refine design,manufacture, and quality control parameters in production offluid-containing devices and processes. The use of such particles isparticularly advantageous for small volume devices, such as micro, nano,and pico volume fluidic, capillary, or deposition instruments, devices,and products.

[0006] These applications of RLS particles relate to the capabilityprovided by such particles to obtain detailed information associatedwith micro-scale, nano-scale, and pico-scale flow, as well as staticproperties or parameters, using appropriately formulated RLS particles.Such information can be readily obtained using simple instrumentationfor detection, or even using detection by eye with appropriatemagnification, though more complex apparatus, especially those havingelectronic analysis and/or control capabilities are advantageous in manyapplications.

[0007] Using such RLS particles, it is possible to detect lightscattering from the particles continuously, without the bleachingexperienced with fluorophores, and with very high sensitivity and signalstability. Detection can even be readily performed on single particles.

[0008] Thus, in a first aspect, the invention provides a method fordetermination of a dynamic property of a fluid volume. The dynamicproperty is determined by determining the distribution or location orboth of at least one light scattering particle using detection of lightscattered from the particle or particles in at least a portion of saidfluid volume. For some devices it may be useful to view the particles inthe entire device simultaneously, while in other cases, it may be usefulto view only a portion of the device, e.g., a valve, flow channel, ormixing chamber. In still other cases, it may be advantageous to follow aparticle or set of particles as they are transported through a device orportion of a device.

[0009] A variety of flow properties can be involved, and thedetermination can involve one or more than one such property, determinedsimultaneously or sequentially. For example, the dynamic property may beflow rate, or particle distribution. In certain embodiments, theparticles are directly or indirectly attached to a biological moleculesuch as a nucleic acid molecule such as nucleic acid probe, apolypeptide such as an antibody or antibody fragment, a lectin, acarbohydrate, or a cell. Thus, the attached light scattering particleprovides a method for determining the distribution of biologicalmolecule or cell in a volume or on a surface. In preferred embodiments,the device is an array, or other device on which molecules are depositedor bound in a localized manner, e.g., spotted on a solid phase surfaceor deposited in a well.

[0010] Similarly, in preferred embodiments, the dynamic property isuniformity (or lack of uniformity) of drying on a solid surface. This isparticularly applicable to development and quality control of arrays,e.g., nucleic acid or polypeptide arrays. Uniformity (or lack ofuniformity) can be uniformity across discrete areas, where the areas areevaluated individually (though the individual evaluations can then becompared), thereby providing a larger scale evaluation or comparison).Examples of such discrete areas are the individual features on a planararray. Alternatively, the uniformity can be evaluated across multiplediscrete areas. As an example, the number of functional probes bound inthe various features on a planar array can be evaluated. In either typeof evaluation, lack of uniformity is typically shown by a pattern to theparticle distribution. Such a pattern may, for example, be irregulardifferences in concentration or density of RLS particle, or a gradientin such concentration or density across an area or volume. In suchembodiments, the uniformity can be evaluated continuously, or atdiscrete times, or at endpoint, or in combinations.

[0011] In preferred embodiment, the deposited volume and/or number offeatures is as described for embodiments of other aspects involvingarrays herein.

[0012] In another example, the dynamic property is a flow pattern in adevice or portion of a device. For example, the device may be amulti-channel device. Such a flow pattern, can for example, be thedistribution of particles across a flow channel, presence and/or size ofeddies or turbulence zones, flow velocity in a portion of a device, or aflow velocity profile across a channel, chamber, or other deviceportion.

[0013] In yet another example, the dynamic property is fluid mixing. Thefluid mixing may be evaluated in one or more portions or elements of adevice, or throughout the entire device. For example, the portion may,for example, be a mixing chamber, a port, a flow channel, a pump, or aflow channel intersection or junction. Such fluid mixing (as well asflow patterns and other properties or parameters) can be evaluated as afunction of device parameters and/or other process parameters. Suchother process parameters include, for example, fluid type (e.g.,identity of solvent), electrical conductivity of the fluid, presence orabsence or amount of one or more dissolved or suspended species, andviscosity of the fluid.

[0014] In preferred embodiments, the device is a small volume device,which may, for example, be a micro volume device such as a microchanneldevice; a nano volume device; a pico volume device; an array chip,plate, or slide; a pump, a port; a valve; a spotting pin; a channeljunction, or a jet head.

[0015] Device and process evaluations can also be performed in devicesor portions of multiple devices that have fluid connection. In this way,interactions between devices in a system can be determined and adjustedin beneficial ways.

[0016] In another aspect, the invention provides a method for analyzingdeposition characteristics of features (spots) on an array, or othersurface arrangement. The method involves depositing at least one fluidvolume on a portion of a solid substrate, where the fluid volumecontains a plurality of light scattering particles, and detecting thelight scattering particles by detecting light scattered from theparticles. The distribution or number or both of the particles isindicative of the deposition characteristics. Alternatively, anothermolecule(s) can be deposited on the array or other surfaces to whichlight scattering particles can be directly or indirectly bound. Then thepresence, amount, and/or distribution of the light scattering particlesserves as an indicator of the presence, amount, and/or distribution ofthe deposited molecule(s). The determinations can also involve otherdeposition characteristics.

[0017] Preferably the array contains at least 5, 10, 50, 100, 200, 500,1000, 5000, 10000 spots, or even more. In particular embodiments, thenumber of features on the array is within a range between any two of thefeature numbers just provided, inclusive of the endpoints.

[0018] Various deposition characteristics of array spots or other spotson a solid phase may be evaluated using the present method. For example,the deposition characteristic can be uniformity (or non-uniformity) ofdeposition. The uniformity can, for example, be evaluated as a2-dimensional distribution of particles within a spot or spots, or as adeposition volume, and/or the uniformity of particle number in depositedspots. The distribution of particles can, for example, be used as anindicator of the distribution of probe molecules in a deposited spot orspots. Likewise, the deposition characteristic can be a drying pattern,for example the distribution of probe molecules during drying of thespot or spots, or a distribution of an active moiety during and/or afterpost-spotting processing.

[0019] The determination of number and/or distribution of a molecule orother detectable item can involve assay of number and/or distribution offunctional components. For example, target, probe, or other bindingmolecule (e.g., an antibody or oligonucleotide probe) can be depositedon a surface. Then a binding assay can be performed, utilizing lightscattering particles as described, to determine the number and/ordistribution of probe (or other) molecules that have a particularbinding function (e.g., specific binding and/or binding strength). Suchassays can likewise be used to determine binding kinetics, e.g., bymonitoring binding on a surface over time and/or binding stability(e.g., by using conditions of a particular stringency, or a range ofstringencies). Monitoring over time can, for example, be performed usingtime lapse or video techniques. In preferred embodiments, the depositioncharacteristic determined is, or is indicative of, functional binding,for example, in nucleic acid hybridization, protein-protein interaction,and ligand-receptor binding.

[0020] As used herein, the term “array” refers to a device having asolid phase surface which has a plurality of features in distinct,physical locations, typically separated by blank or empty areas. The“features” are locations where particular molecules, often biomolecules,are immobilized for conducting assays. In most current arrays, theimmobilized molecules are probes that bind to target molecules in asample or samples applied to the array. As used herein, the term “array”is used as a general term for any such device. As used herein, the term“array chip” refers to an array with a planar solid substrate withsurface area of 1 in² or less; the term “array slide” refers to an arraywith a planar solid substrate with a surface area greater than 1 in² upto 4 in² inclusive; the term “array plate” refers to an array with asolid substrate with a generally planar surface. In some embodiments,the plate has depressions, e.g., wells, for containing liquids.

[0021] In connection with arrays, the terms “features” and “spots” areused synonymously. The features may, for example, be particular areas ona flat surface, wells, or channels (which may be oriented in a flat,viewing plane of the array, or endon to a flat, viewing plane of thearray). Preferably the features are particular locations on a flatsurface, e.g., specific oligonucleotide or polypeptide-containing areason a glass or plastic slide or chip.

[0022] In particular embodiments, the small volume deposited at one ormore features is 1 pL to 10 nL, preferably 10 pL to 10 nL, morepreferably 50 pL to 10 nL, still more preferably 50 pL to 1 nL. Inadditional particular embodiments, the small volume deposited is 50 pLto 500 pL, 50 pL to 200 pL, 10 pL to 200 pL, 10 nL to 200 nL, or 200 nLto 2 μL. Also in particular embodiments, the volume deposited is in avolume range described by taking any two different particular volumesjust specified as the inclusive endpoints of the range.

[0023] As in the previous aspect, in preferred embodiments the array hasa t least 5, 10, 50, 100, 200, 500, 1000, 5000, 10,000 features, or evenmore, or the number of features is in a range described by taking any 2different values, as described, as the endpoints of the range.

[0024] The invention also provides a method for analyzing fluid flow inat least a portion of a device, preferably in a small volume device,more preferably in a plurality of portions of a small volume device. Themethod involves inserting a suspension of light scattering particles inthe device, illuminating the light scattering particles in a pluralityof portions of the device, and detecting the presence of lightscattering particles as an indication of the fluid flow. The flow can,for example, be continuous, stopped, or pulsatile flow.

[0025] The portion or device may be any that is appropriate for fluidflow that allows, or can be adapted to allow illumination of particleswithin or on the device or portion of interest. Thus, preferably thedevice has at least a portion that is exposed or transparent to light,preferably to visible wavelength light, preferably the incident lightbeam is provided by laser or collimated incident light beam.

[0026] In connection with flow devices and/or other fluid-containingdevices, light scattering particles can also be used to monitor bindingof molecules in solution to the interior walls of the device. In manycases, such binding is undesirable, for example, as it results in lossof a significant portion of sample or because it makes quantitation ofmaterial or process results problematic or questionable. In preferredembodiments, determination of binding can be carried out similarly tobinding determinations on an exposed surface (e.g., on a microarray).For example, in an exemplary embodiment, the solution containing themolecule of interest is placed in or flowed through the device orportion of a device. Unbound material is washed out of the device orportion, and bound molecules are detected by directly or indirectlybinding light scattering particles to the bound molecules and detectingthe light scattering particles as an indication of the presence of thebound molecules.

[0027] Flow detection and analysis can be performed in various waysdepending on the flow property or properties of interest. For example,time lapse imaging can be used to provide particle images at discretetime points, extended exposures can be used to provide trace linesshowing particle paths, and video images can be used to provide movingparticle images. Combinations and other options can also be used.

[0028] In addition, in another aspect, the invention provides a methodfor analyzing at least one surface characteristic of a solid substrateor porous matrix. The method involve detecting the distribution ornumber or both of the light scattering particles on at least a portionof the substrate, e.g., a surface, by detecting light scattered from theparticles, following treatment of at least a portion of the substratewith at least one fluid volume containing a plurality of lightscattering particles. The distribution or number or both of theparticles is indicative of the characteristic.

[0029] As used herein in connection with suspensions of light scatteringparticles, the terms “treatment”, treating” and words of like importrefer to contacting a material or composition with the suspension, andcan also include additional processes, for example, non-covalentbinding, covalent binding, and washing.

[0030] In the context of substrate analysis, e.g., surface analysis, theterms “characteristic”, “surface characteristic”, and “matrixcharacteristic” refer to a physical, chemical, and/or electricalproperty of the solid substrate, e.g., texture, planarity, porosity,surface charge, surface charge uniformity, hydrophobicity,hydrophilicity, reactivity, and combinations thereof, as well as otherproperties. One of ordinary skill in the art will recognize that thecharacteristics that can be analyzed are those that affect the numberand/or distribution of particles on the surface, either directly orindirectly (e.g., through another component that is attached or becomesattached, directly or indirectly, to the particles).

[0031] Thus, in preferred embodiments, the characteristics analyzedinclude one or more of surface on matrix uniformity, uniformity of oneor more coatings, uniformity of charge, uniformity of hydrophilicity,uniformity of hydrophobicity, and uniformity of charge density. Thecharacteristic can concern a surface or surfaces and/or substratethrough at least a portion of a porous matrix.

[0032] The terms “hydrophobicity” and “hydrophilicity” have their usualtechnical meaning, referring to whether a material does not associatereadily with water, or associates readily with water, respectively.

[0033] Also in preferred embodiments, the solid substrate is a glasssubstrate, a functionalized glass substrate, a plastic substrate, asilicon substrate, a membrane substrate, a metallic substrate, orcombinations thereof.

[0034] The determination of light scattering particles on the surfacecan be performed with illumination and detection on the same side of thesubstrate, e.g., membrane (for either transparent or non-transparentmembranes or other substrates) or from opposite sides (for essentiallytransparent membranes or other substrates, and substrates that can bemade essentially transparent).

[0035] In the context of this invention, “membrane” refers to a thin,flexible impermeable or microporous material, preferably syntheticmaterial. Preferably pores or channels in the membrane are no largerthan 20 μm, more preferably no larger than 10, 5, 2, 1, 0.5, 0.2 or 0.1μm, or in a range specified by any two of these specified endpoints.Preferably, a membrane is a uniform sheet of material with essentiallyuniform composition, e.g., a film, though in some embodiments a membraneis fibrous material, e.g., woven or matted fibrous material. Examples ofcommonly used materials include nylon, nitrocellulose, polyvinylidenefluoride (PVDF), and cellulose.

[0036] As used herein, the term “matrix” or “matrix material” refers toa porous material, preferably a microporous material. A porous materialis one with channels that allow entry or passage of fluid and/or air.Such channels may be discrete or interconnecting, and may be throughchannels and/or blind channels, but are preferably through channels.Preferably such passages are of sufficient size to allow entry orpassage of light scattering particles of at least 1 nm diameter, morepreferably at least 10, 20, 30, 40, 60, 80, 100, 120, or 150 nm.Preferably the channels are, on average, at least 50, 100, 200, 400,600, 800, or 1000 nm in cross-section. The degree of porosity can vary,e.g., representing at least 1, 2, 5, 10, 20, 40, or 50% or even more ofa surface of a material. Thus, matrix materials include such exemplarymaterials and items as membrane filters, fibrous filters, and sinteredglass filters.

[0037] The term “functionalized” refers to a chemical modification thatprepares a material, e.g., a glass, plastic or metal surface forsubsequent chemical interaction by attaching or creating suitablefunctional groups or moieties. Thus, for example, an analysis candetermine the number and/or density of accessible groups on a surfaceafter functionalization.

[0038] As used herein, the term “fluid” refers to a material orcombination of materials that is liquid under the relevant pressure andtemperature conditions, e.g., at room temperature and one atmosphere.

[0039] In the context of this invention, the term “device” means anarticle of manufacture that includes one or more channels or reservoirsor locations for fluid to be present, e.g., for flow or deposition.

[0040] The term “microchannel” refers to a channel of sufficient size toallow fluid flow, preferably a channel generally in the form of a tube,that has mean cross-sectional measurement of 3 mm or less. Inparticular, embodiments the channel is 2 mm or 1 mm or less, or 0.5 mmor less, or 100 μm or less, preferably 10 μm or less or 100 μm or less,still more preferably 80 nm or less, or 60 nm or less, or 40 nm or less,or 20 nm or less.

[0041] “Microscale” refers to devices or portions of devices orprocesses with dimensions of 3 mm or less, preferably 1000 μm or less,generally in the range of 1-500 μm, for functional parts or processes.Thus, channels, junctions and the like are typically of such dimensions.

[0042] “Nanoscale” refers to devices or portions of devices or processeswith dimensions of 1000 nm or less, generally in the range of 1-500 nm,for functional parts or processes. Thus, channels, junctions, and thelike are typically of such dimensions.

[0043] “Microfluid dynamics” refers to the fluid dynamics in microscalesystems, preferably in systems with flow channel dimensions of 1 mm orless, 500 μm or less, 100 μm or less, 500 nm or less, 100 rim or less,50 nm or less, or even smaller. Thus the term refers to the fluidbehavior in such channels.

[0044] The term “subfluidic region” refers to a portion of a flowchannel or reservoir. Generally the term is used in connection with thebehavior of fluids in such a sub-region in connection with microscale ornanoscale processes or channels or reservoirs. Likewise, the term“sub-flow pattern” refers to the fluid flow pattern or behavior in asub-region of a flow channel or reservoir, generally microscale ornanoscale. Such regions and flows can be monitored using the methods ofthe present invention.

[0045] “Microfabrication” refers to the techniques and processes ofproducing a microscale or nanoscale device. Exemplary techniques includephotoetching, laser shaping, micromachining, and the like. The methodutilized will depend on factors such as the scale and the materialsbeing utilized.

[0046] The term “fluid deposition” refers to the process of placing avolume, generally a small volume, on a solid phase surface. Thedeposition may, for example, be on a flat surface, or in a depression,or cavity in the surface, or on the walls of a tube through a solidphase material. Exemplary methods include those commonly used forproducing microarrays. A variety of such deposition methods, as well asother factors in production of arrays, are described in MicroarrayBiochip Technology, Mark Schena, ed., Eaton Publishing, Nattick, Mass.,2000, and are applicable for the present methods. Deposition methodsinclude, for example, piezoelectric, inkjet, solenoid/piston, and pinspotting. Deposited volumes are preferably 1 μL or less, 0.5 μL or less,0.1 μL or less, or more preferably 10 nL (nanoliters) or 1 nL or less,or even 500 pL (picoliters) or less, 200 pL or less, 100 pL or less, or50 pL or less.

[0047] The term “small volume” as used herein refers to a volume of 10mL or less, preferably 5 mL or less or 1, 0.5, 0.1 mL or less, morepreferably 10 μL or less, or 1, 0.5, 0.1 μL or less, still morepreferably 10 nL, 1 nL, 0.5 nL, or 0.1 nL or less. Still smaller volumesare also included. Thus, a small-volume device has a volume within thedevice within the limits described. The term may also be used inconnection with portions of a device, or a process, or sub-process.

[0048] “Microvolume” refers to volumes of equal to or less than 1000 μl,generally in the range of 1 to 1000 μl; in exemplary embodiments100-1000 μl, 200-800 μl, 100-500 μl, 1-500 μl, 800 μl or less, 500 μl orless, or 200 μl or less.

[0049] “Nanovolume” refers to volumes of 1000 nL or less, generally inthe range of 1 to 1000 nL, in exemplary embodiments 1-600 nL, 1-400 nL,100-1000 nL, 100-600 nL, 800 nL or less, 500 nL or less, or 200 nL orless.

[0050] “Picovolume” refers to volumes of 1000 pL or less, generally inthe range of 1 to 1000 pL, in exemplary embodiments, ₁₋₆₀₀ pL, 1-400 pL,100-1000 pL, 100-600 pL, 800 pL or less, 500 pL or less, or 200 pL orless.

[0051] “Resonance light scattering particles” (RLS particles) refers toparticles that elastically scatter incident light with high efficiency.Preferably the particles are metal or metal-like particles. Preferredexamples include gold particles, silver particles, and mixed compositiongold and silver particles as well as particles containing at least 1, 5,10, 25, 50, or 5% by weight of gold or silver or a combination of goldand silver. Examples of mixed composition particles are particles withsilver surrounding a gold core, and gold over silver. For silverparticles, a thin outer layer of gold can be advantageous, e.g. byproviding light scattering characteristics of a silver particle whilestabilizing the particle with the gold outer layer. Particles are alsoincluded that contain or are composed of other materials that havesufficient light scattering intensity to allow use as labels or as flowmarkers, preferably with particles sizes of 1-500 nm.

[0052] The size of RLS particles can be selected as needed or useful forparticular applications. For example, particles can be selected toprovide different colors on scattering of white, or more generallypolychromatic light. Likewise, it may be beneficial to select particlesof a particular sizes or with certain size limitations based on thedimensions of the fluid-containing portions of the device. In manycases, it is preferable to utilize particles that are small compared tothe dimensions of the fluid channel or chamber, e.g., ≦⅕, {fraction(1/10)}, or {fraction (1/20)}th. In many applications such sizelimitations are useful so that that particles will move freely throughthe device, and preferably will respond to eddies, turbulence, and othersub-features of the flow in the device to allow determination of thosecharacteristics.

[0053] By “metal-like” particles is meant any particle or particle-likesubstance that is composed of metal, metal compounds, metal oxides,semiconductor (SC), superconductor, or a particle that is composed of amixed composition containing at least 0.1% by weight of metal, metalcompound, metal oxide, semiconductor, or superconductor material.

[0054] By “coated” particle is meant a particle has on its surface alayer of additional material. The layer is there to chemically stabilizethe particle in different environments, and/or to bind specificmolecules by molecular recognition means. Such coatings include, forexample, inorganic and organic compounds, polymers, proteins, peptides,hormones, antibodies, nucleic acids, receptors, and the like. Asdescribed in the Yguerabide references cited herein, coated metal-likeparticles have similar light scattering properties as compared touncoated metal-like particles, both of which have superior lightscattering properties as compared to non-metal-like particles.

[0055] By “non-metal-like” particles is meant particles that are notcomposed of metal, metal compounds, superconductor, metal oxides,semiconductor, or mixed compositions that are not composed of at least0.1% by weight of metal, metal compound, metal oxide, superconductor, orsemiconductor material.

[0056] In this invention, it may be advantageous to provide a pluralityof different particles, where each of the plurality is separatelydistinguishable. The plurality of different particles means that thereis one or more individual particles, generally a large number ofindividual particles, of each of the different, separatelydistinguishable particles. The composition, size, and shape of theparticles are chosen to provide distinguishable light scatteringparticles, e.g., different colors and/or different intensities. Forexample, roughly spherical gold particles of 40, 60, and 80 nm diametercan be used to provide distinguishable colors when illuminated withwhite (or polychromatic) light. In particular embodiments, 2, 3, 4, 5,6, or even more distinguishable particles are used. The plurality ofdifferent particles can, for example, be used to analyze mixing offluids from two different sources, e.g., from two different channelswithin a device, or to visualize the mixing of a small volume as it iscombined with a larger volume. Such different particles can be provided,for example, using gold particles of differerit sizes, and/or silverparticles of suitable size to provide distinguishable colors (e.g., twoor more of 40, 60, and 80 nm gold particles, and 40 nm silver particles.Those skilled in the art will recognize a variety of other applicationsfor multiple distinguishable particles.

[0057] Also, non-spherical particles may be used to provide usefulinformation on flow or fluid properties, e.g., flow rate, viscosity,turbulence, flow gradients, and the like. Non-spherical particles thatare elongated, e.g., particles that are generally oval or rodshaped, canbe distinguished from generally spherical particles by flickering in thescattered light as they rotate. Thus, the observable flickering willcorrelate with the flow properties, such as those listed above, e.g., byreduction in the flicker rate as viscosity increases. Thus, suchelongated particles will provide additional characterization of flowproperties in a system or device.

[0058] Preferably, but not necessarily, the detection of lightscattering in the present methods is performed using simpleinstrumentation. By simple instrumentation is meant with magnificationless than 500×, and without confocal imaging, and preferably without useof laser illumination. However, in some embodiments, laser illuminationis advantageous, as laser may have important applications in providingincident light precision. Use of laser illumination can readily be usedwith sets of particles selected to provide multiple distinguishablecharacteristics, e.g., distinguishable intensities, distinguishing 2 ormore different particles. Typically the sets of particles are selectedby size and/or composition to provide the distinguishablecharacteristics. However, in some embodiments, it will be beneficial touse such apparatus, and/or to use electronic imaging devices. Inaddition, electronic image processing and analysis tools can also beused.

[0059] The term “dynamic property” refers to a property orcharacteristic of a system or material that changes over time. Theproperty may, however, reach an endpoint. Examples include, withoutlimitation, flow rate, mixing, distribution of a material, distributionof material during drying, and binding, distribution and/or number offunction molecules or components, stability of material on a surface(e.g., as a function of experimental processing), presence of flowfeatures such as turbulence or micro eddies or other extremely localflow dynamic effects. In particular embodiments, one or more of thesedynamic properties is determined, at one or more timepoints orcontinuously over a time interval(s)

[0060] Detection may be performed using any of a variety of differentdetectors. One of ordinary skill in the art will be familiar withnumerous detectors. For example, in some applications it may besufficient for the particles to be viewed by eye. However, in otherapplications it may be preferable to use a film or electronic detector(e.g., a film or electronic camera), that produces a picture and/orelectronic record. Such cameras may be still (which may be used intime-lapse manner) or video cameras. The film or electronic image can befurther processed and/or analyzed to identify features (e.g., the numberand/or position of particles) and/or to characterize one or moreproperties of such features. Of course, video cameras may be used withframe-grabbers to allow single or multiple image processing and/oranalysis. Electronic cameras include, for example, charge coupled device(CCD) cameras, charge injection device (CID) cameras, and ComplementaryMetal Oxide Semiconductor (CMOS) cameras.

[0061] While the description of aspects and embodiments herein isgenerally presented in terms of fluids (used herein as equivalent to theterm liquids), the analysis of flow behavior using RLS particles canalso be performed in gaseous flow, e.g., air flow. Typically, but notnecessarily, the velocities in such flow are greater than for liquids.Also typically, RLS particles used for gas flow analysis will be small,e.g., 1-40 nm, preferably 1-20 or 1-10 nm. In some applications it isalso beneficial to use particles of lower density than solid metalparticles. Examples include particles with a metal shell over a lowdensity core, thereby maintaining high light scattering intensity butenhancing the ability of the particle to remain suspended in the gas fora useful period of time. The detection and analysis of particledistribution, flow pattern, and other properties in gas systems isessentially the same as for the fluidic systems described herein, andare part of the present invention.

[0062] Additional aspects and embodiments will be apparent from thefollowing Description of the Preferred Embodiments and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0063]FIG. 1 is a schematic illustration of a system arranged fordetecting light scattering particles in a flow device using a narrowdetection field, with electronic detection and data acquisition, andwith a computer for data storage and/or data processing.

[0064]FIG. 2 is a schematic illustration of a system arranged fordetecting light scattering particles in a flow device using a wide angledetection field, with electronic detection and data acquisition, andwith a computer for data storage and data processing.

[0065]FIG. 3 shows four sequential images of light scattering particlesflowing through a microchannel using dark field trans-illumination.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] The present invention concerns the use of resonance lightscattering (RLS) particles in connection with small volume devices andprocesses, e.g., in the design, manufacture, and quality control ofsmall volume devices and instruments. Individual RLS particles ofappropriate size and composition can be detected using simpleinstrumentation, or by eye with appropriate magnification. Thiscapability provides many applications for the use of RLS particles inindustrial production of micro and nano volume fluidic, capillary ordeposition instruments, devices and other products.

[0067] In particular, this invention concerns the ability to obtaindetailed and specific information associated with micro-scale,nano-scale, and pico-scale flow and suspended and attached materialdistribution using appropriately formulated RLS particles, even withsimple instrumentation.

[0068] Current methods for such analyses typically rely upon detectionof fluorescent or chromogenic dyes using a time-lapse or high-speedvideo device. In flow systems, a fine stream of the detection agent maybe used for introduction into the system. However, information obtainedusing these methods is of limited utility with respect to a significantarea of the flow channel because of limited sensitivity and rapiddiffusion and dilution of the detected agent.

[0069] In contrast RLS particles are highly intense and particulate innature. Using properly formulated RLS particles under essentially thesame conditions, one can observe and measure flow patterns, e.g.,micro-flow and nano-flow patterns, under various experimental andapplied run conditions over extended distance and time, as well asbinding properties, deposition characteristics, and other properties.

[0070] In micro-, nano-, and pico-scale deposition applications, RLSparticles possess properties that provide important advantages overcurrent methods that largely rely upon fluorophores or isotopes. Inthese methods, the inability of fluorescent dyes to maintain a strong,non-diffuse, and consistent signal limits the utility of these detectionagents. In contrast, RLS particles provide an important tool forobservation and measurement of fluid distribution and uniformity duringdeposition and drying at high resolution. RLS particles also providebetter precision and sub-fluidic resolution. Again, simpleinstrumentation, such as a conventional laboratory microscope unit withtime lapse video may be used. Many other instrument configurations canalso be used.

[0071] Thus, RLS particles can be formulated for and used in anyapplication in which direct visualization, and qualitative and/orquantitative measurement of distribution or flow properties of smallliquid volumes and/or suspended or attached components is desired. Theinherent stable intensity and particulate nature of RLS particles makethem ideal for such applications as they provide a quantifiable(individual particle counting and/or integrated intensity), stablesignal that does not diffuse. Another aspect of the invention is theproduction and formulation of RLS particles for optimal performance in aparticular micro-volume deposition or flow application. Exemplaryapplications of these particles are provided below, but are not intendedto be limiting to the invention.

[0072] While RLS particles are particularly useful for very small volumedevices, such particles can also be used in connection with largervolume devices and processes, e.g., in the design and testing of largerpumps and other flow system components.

[0073] As indicated above, Yguerabide at al., PCT/US97/06584, Yguerabideet al., PCT/US98/23160, and Yguerabide et al, U.S. Pat. No. 6,214,560all describe a large number of different options for particles, particlecoatings, binding of molecules to particles, and method and apparatusfor detecting particles by light scattering. All of the informationprovided therein can be utilized in embodiments of the presentinvention.

[0074] Preparation of RLS Particles

[0075] A large number of different methods for preparing lightscattering particles of different compositions and different sizes havebeen described and can be used in the present methods. Examples of suchmethods are provided in Yguerabide PCT/US98/23160, WO 99/20789; Frens,Nature Physical Science 241: January (1973); Frens, et al., Kolloid-Z.u. Z. Polymere 250:736-741 (1972); Turkevitch, et al., Diss. FaradaySoc. 11:55-75 (1951); and Stevenson, “Some Experiments on ColloidalGold”, Ph.D. Thesis, Princeton University (1949). Exemplary preferredparticles are gold and silver particles, or particles composed of goldand silver, or gold and silver in combination with one or more othermetals and/or one or more other materials. Particles can also be sizefractionated to provide particular size ranges for particles. Thisfractionation is advantageous, for example, for discrimination ofdifferent size particles.

[0076] For example, a particle growing procedure can be utilized thatinvolves first making a preparation of “seed” gold particles which isthen followed by taking the “seed” particle preparation and “growing”different size gold (see Examples 1 and 4) or silver particles (seeExample 3) by chemical methods. For example, 16 nm diameter goldparticles are used as the “seed” particles and larger diameter goldparticles are made by adding the appropriate reagents (see Example 4).This is also useful for making mixed-composition particles.

[0077] It is also advantageous to stabilize the particles, e.g., againstclumping or precipitation. This can be done, for example, by coating theparticles with another substance that enhances their stability. Avariety of such coatings are described in the Yguerabide applicationsreferenced above. Coating in the present case may also reduce oreliminate non-specific particle binding to the micro or nano flow devicesurfaces in cases where that is advantageous.

[0078] While gold particles can advantageously be used in the presentmethods, other types of RLS particles can also be used. For example,silver particles provide intense light scattering and are used inembodiments of the present invention.

[0079] Detection of Light Scattering Particles

[0080] For the applications of this invention, light scatteringparticles are commonly detected in solution, although the detection canalso include particles bound on a surface, e.g., for determination ofarray spot or feature drying characteristics or binding distributions.Suitable apparatus for such detection can be of many different types andconfigurations. A variety of useful detection methods are described, forexample, in Yguerabide at al., PCT/US97/06584, WO 97/40181; Yguerabideet al., PCT/US98/23160, WO 99/20789; Yguerabide et al, U.S. Pat. No.6,214,560; and U.S. Application Yguerabide et al., 08/953,713. Ingeneral, a variety of different light sources can be used, for example,incandescent lamps, light emitting diodes, lasers, polarized lightsources (e.g., linearly polarized), continuous sources, and pulsedsources.

[0081] The illumination beam is directed toward the light scatteringparticles to be detected. The light can be transmitted directly or canbe guided through a light guide, such as a prism or optical fiber.

[0082] The light scattered from the particles can be detected in variousways, including, for example, by unaided eye, by eye through a lightmicroscope (e.g., using magnification of 1-100×, 50-200×, 100-500×, or500-1000×), with a still camera (analog or digital), with a videocamera, with a photodiode or photodiode array, or with one or morephotomultiplier tubes, or with combinations thereof.

[0083] While the brightness of the light scattered from particles suchas gold or silver particles is sufficiently intense to allow detectionunder a wide range of conditions, including conditions with much morebackground light than optimal, it is preferable to arrange theillumination and detection apparatus to minimize background light. Thus,in order to increase sensitivity of detection, preferably techniquesdescribed in the Yguerabide et al. patent and applications cited aboveare utilized. For example, preferably the detector is positioned toreduce the amount of reflected light that enters the light collectioncomponents. Preferably the illumination source and detector are arrangedto avoid detection at the direct illumination beam and to minimizedetection of reflected and refracted light. Other exemplary techniquesinclude the use of spatial filters to reduce stray light, band-passfilters to enhance sensitivity, use of laser or highly collimated lightsources, and use of refractive index matching or other refractive indexenhancement techniques. Such refractive index enhancement methodsinvolve the selection or manipulation of the refractive index of mediacontaining or covering the particles, or otherwise covering a surface orsurfaces that would otherwise contribute to background light scattering,to reduce background light scattering and/or to enhance the lightscattering from the particles used for detection (and/or to reduce thelight scattering from other system components such as other particles insuspension).

[0084] Two different exemplary methods and associated apparatus aredescribed here that can be selected depending on the design of thedevice to be monitored. If the device accommodates a narrowing of theflow into a channel of <10 μm or so, a simple detection system composedof a collimated light source and an area sensor such as aphotomultiplier tube or PMT, or phototransistor. The detection sensor ispreferably placed behind a lens system to focus the scattered light onthe surface of the detector. The entire detection system is placed at anangle to the light source so as to create a darkfield illuminationcondition, thereby keeping unwanted light from entering the collectionoptics. (See, e.g., FIG. 1). The angle of both the illumination anddetection are preferably optimized to achieve the maximum scatteringsignal from the particles, with minimum background contribution from theillumination. In this approach, an average signal is delivered to aninterfaced computer of the entire field of view containing individual ormultiple particles. The intensity of the signal from the detector isdirectly proportional to the number of particles in the field of view(assuming the system is operated within the dynamic range of thedetection system). In order to calculate particles per unit volume in adynamic system with continuous flow, the flow rate is controlled andfactored with the detection signal to calculate particles per unit givenvolume of flow.

[0085] A second embodiment is particularly applicable to devices inwhich a narrowing of the flow path was not possible or if more accuratecounting were desired. (See, e.g., FIG. 2) In this design, theillumination is a broader beam, e.g., across the entire channel. A highframe rate camera and lens is positioned over the channel. For coverageof the entire channel, the limit on channel width is dependant on theresolution of the camera and the size of the particle. Softwareprocesses the streaming images, counting and/or tracking individualparticles from the point of entry into the camera frame until they moveout of view. CMOS camera sensors are being produced with very high framerates and built-in logic for detection and quantitation of movingobjects in the field of view. Such cameras are suitable for the presentuse, but other cameras can also be utilized. Illumination is configuredin a darkfield arrangement similar to the previous embodiment. Withsuitably sized view fields, the detection is not limited to a singlechannel, but can cover a larger portion of a device, even included anentire device.

[0086] Of course, many other configurations can be constructed andutilized, depending on the particular application. Examples includearrangements for detecting RLS particles at multiple view planes (e.g.,2 or 3 parallel view planes at different depths in a fluid), detectingfrom multiple directions (e.g., 2 or 3 dimensions), tracking particlesthrough as device by moving the device in the field of view or movingthe field of view over the device (e.g., to maintain particularparticles within the field of view), and detecting particles of multiplelocations within a device or in connected devices (e.g., 2, 3, 4, ormore locations), as well as combinations of these.

[0087] Microfluidic Systems

[0088] RLS particles can be used for many applications with microfluidicsystems and devices. Some device examples are reviewed in Chemical &Engineering News, 77(8), 27-36, 1999. Further examples are well-known tothose of ordinary skill in the art. Such devices are used for a varietyof “lab on a chip” applications, among others, including drug screening,diagnostics, chromatography, and microeletrical field flowfractionation. Exemplary, non-limiting uses of RLS particles inmicrofluidic systems are briefly described below.

[0089] Microfluidic devices can be constructed in a variety of ways. Oneexample is to use a micromachining process involving etching of asilicon substrate. The etched silicon can be sealed with glass tocomplete the channels and chambers. The fluidic connections can then bemade at the edges of the device. Typically in such devices, flowchannels are greater than 5 μm wide and elements are 0.5 μm to 200 μmdeep.

[0090] Other methods of manufacture of microfluidic devices involvephotolithography of a thin plastic layer deposited onto a glass orsilicon substrate. In yet another technique, plastic injection moldedparts can be fabricated with very small channels, and assembled intomicrofluidics devices using ultrasonic or solvent welding techniques.

[0091] Commonly, flow in microfluidics devices is low Reynolds numberflow, such that R_(e) is much less than one. The result is that there isoften little turbulence. However, with such small dimensions, diffusionbecomes a very significant factor. Still, an advantage offered bymicrofluidics is that, due to the very small fluid volumes involved andthe resulting low inertias, flow can be reversed, or otherwise switchedvery fast. For example, flow can be switched at a junction by changingthe applied fluid pressure, with the switching having been accomplishedin as little as 20 msec. A potential complication is that microfluidicdevices have low volume/surface ratios. Consequently, the ratio ofmolecules in solution to molecules adhering to a surface can besubstantially lower than in large volume devices. If this is found tocreate difficulties, it can be addressed by coating the particles and/orthe device surface to reduce the interaction.

[0092] Micro Fluid Dynamics

[0093] RLS particles allow one to directly measure individual andspecified grouped sub-fluidic regions and components that function inmicrofluidic devices. Practical application of fluid dynamics inmicroflow environments has indicated an important balance between flowrate, flow pressure, fluid viscosity, channel dimension, channelmaterial, and channel friction as important for optimal performance. Indesign and testing of any micro fluidic system, the ability to obtainvivid, real time measurement of flow and sub-flow patterns throughoutthe entire micro-channel system offers a tremendous advantage. For suchanalysis, current methods must treat regions of individualmicro-channels as independent units instead of as components in anintegrated system. As an example of the advantages provided by thepresent invention, flow dynamics in one micro-channel junction can beexplored in conjunction with the flow dynamics of a feeder valve severalchannels away. Similarly, RLS particles can be used to design and testchannel junctions and channel design configurations to optimize mixingor distribution of reaction components. Here again, RLS particles offeran advantage over existing approaches for obtaining useful informationon component interaction, dynamics, and mixing across an integratedfluidic system.

[0094] Use of RLS Particles for Design and QC of MicrofluidicChannels/Devices

[0095] Given their intense and non-diffusing signal generating power,RLS particles provide an excellent tool for guiding the design andperforming quality control of microfluidic channels and associateddevices. RLS particles used for these applications offer superior andquantifiable measurement of localized fluid dynamics in comparison tofluorescent dyes and other typically used fluid-dynamic label systems.In this application, RLS particles are introduced into microfluidicchannels and hydrodynamic flow is visually monitored using appropriatelyconfigured illumination and detection optic instrumentation andsoftware. Flow rates and microflow patterns can be determined bymeasuring and/or visualizing the particle flux across desired channelvolumes, areas or dimensions.

[0096] Thus, RLS particles can be used to optimize channel design andmanufacturing processes to meet the fluid dynamic requirements of themicrofluidic device. This approach is not limited to analysis of variousmicrofluidic channel designs per se, as flow properties of otherassociated flow device elements not limited to pumps, injection ports,valves, channel junctions and mixing stations can be similarlyinvestigated. In addition, one can use RLS particles to study laminarfluid dynamics or microchannel surfaces of various character includingfabricated or deposited electrode materials or other surface coatings.

[0097] In addition or in combination with evaluating flowcharacteristics, e.g., evaluating the effects of changes to the shapeand/or material of the device, the present RLS particle methods can beused to evaluate the effects of fluid characteristics, either alone orin combination with evaluating device parameters. In particularapplications, this is useful for microchannel devices. Such fluidcharacteristics can, for example, include viscosity, solvent, electricalconductivity, identity of dissolved molecules, and pressure, andcombinations of these. As an example, the dispersion of a sample volumeor reagent volume can be monitored as a function of sample (or reagentsolution) viscosity and/or bulk solution viscosity. Likewise, in bindingapplications, the binding kinetics can be monitored as a function ofsolution viscosity. Monitoring both fluid characteristic and devicecharacteristic effects allows co-optimization of a system, oridentification of advantageous compromises, e.g., compromises in designand process that allow extended operating range. Many other specificapplications will be apparent to one of ordinary skill in the art.

[0098] Microfabrication

[0099] RLS particles can be used as tools to aid microfabricationmethods for the design, production, testing, and quality control ofmicrofluidic devices and systems. For example, using RLS particles onecan study and optimize an entire micro fluidic system, including, forexample, microcirculation devices, micropumps, micropump controllers,microchannel sensors, microsensors and associated actuators,microchambers, microelectrodes, and the like, as an integrated system.This offers distinct advantages for system design and testing. Theseapplications may include optimization of methods for design,engineering, manufacture, and/or testing microchannels. RLS particlesallow one to rapidly design, product, and test microchannels made in anyof various ways and various dimensions, if derived, rapidly andquantitatively.

[0100] For particular microflow applications, RLS particles can furtherbe used to optimize production processes and flow properties ofmicrochannels coated with various components. For example, in “lab on achip” applications, RLS particles can be used to rapidly testformulations, methods, and performance of coatings that affectparticular steps, such as binding or release of molecules in an analysisprocess. RLS particles can also be used for the design, testing, andproduction of “flow-through” chips that feature specifically immobilizedprobes, for example, in porous silicon wafers. Refractive index matchingis preferably used to reduce background scattered light in suchapplications.

[0101] Fluid Deposition Systems

[0102] RLS particles can be used for detailed study of the propertiesand dynamics of small liquid volumes on solid surfaces. Whereas this isparticularly the case in the rapidly growing field of microarrayanalysis, the use of RLS particles can be applied to other solid phasesystems. This invention provides applications to microarrays althoughuse of RLS particles to study properties and dynamics of small liquidvolumes on solid surfaces is not limited to array applications.

[0103] Exemplary Description of how RLS Particles are Used forDetermining Array Spotting Formulations and Processes

[0104] The intense signal obtained from both individual RLS particlesand a population of RLS particles on a surface, when observed andmeasured as integrated intensity, enables one to visually determine thedistribution and homogeneity of deposited nucleic acids or proteins withan unprecedented level of ease, precision and resolution.

[0105] Experimentally, for example, one can determine the surfacedistribution of a deposited biomolecule on a glass slide, polystyrene orother plastic surface, or on membrane substrates such as nitrocellulose,PVDF or nylon by the following steps: Deposit and bind thebiomolecule(s) to the surface or substrate, treat the surface orsubstrate with a blocking agent to prevent non-specific binding of RLSparticles, react appropriately derivatized RLS particles with thesurface or substrate under conditions that affect specific binding ofthe RLS particles to the deposited biomolecules, wash away RLS particlesthat are not specifically bound to the deposited biomolecules, andmeasure the morphology and surface distribution properties of thedeposited biomolecules on the surface or substrate under appropriate RLSillumination and detection conditions. Membrane substrates in thesystems above are generally made transparent by refractive indexmatching or other methods prior to viewing or quantitating.

[0106] In addition, one can also determine the distribution of the RLSsignal in a functional test wherein the prepared slide or substrate hasexperimentally undergone defined hybridization, antibody binding,ligand-receptor binding, or other binding and wash conditions. In thiscase, monitoring the distribution of RLS signal provides a means todetermine the distribution of functional biomolecules (i.e. biomoleculesdeposited in a manner that facilitates and permits stable cognate targetbinding) within the defined array feature.

[0107] Examples of this approach are provided below.

[0108] Microarrays

[0109] Microarray technology has revolutionized the analysis of geneexpression and DNA sequence and protein detection and association byallowing one to analyze the binding of biomolecules present in a samplein a highly multi-parallel manner. Microarrays have become the centraltool to explore the function of the genome and understand biologicalfunction. Large sets of individual proteins or gene probes are “spotted”onto solid surfaces and interrogated with analytes (targets) in aprepared sample. Quantitation of target binding data obtained allows oneto acquire a tremendous amount of information for a large number ofgenes simultaneously. This approach has greatly accelerated the studyand understanding of biological systems.

[0110] Currently, several methods are widely employed to deposit nucleicacid or protein onto solid surfaces in production of microarrays,including pin transfer, syringe-solenoid and piezoelectric. Thesemethods generally transfer between 250 and 10 nL per spot, althoughsmaller volumes are possible (and would probably be more widely used ifnot for limited detection sensitivity and detector resolutionlimitations) with the latter two methods. In all cases, a small volumeof fluid containing the desired probe is applied to the surface.

[0111] Surfaces for deposition are often derivatized glass microscopeslides, although other substrates, including plastic or membranesurfaces have also been used. Once deposited, the small liquid volumegenerally dries on the surface over a brief period of time. In somecases, application of the small liquid volume containing the desiredprobe takes place in a controlled humidity environment. This allows thetarget to interact more extensively with the glass substrate byremaining for a longer time in the liquid state. The method of spottingshould generally be developed in concert with binding and detectionaspects of the array system. Current methods to examine deposition ofsmall liquid volumes on arrays include isotopic (generally ³³P or ³²P)or fluorescence, however both methods are limited or less desiredbecause of isotope disposal issues and/or poor analytical resolution,sensitivity, and/or signal stability.

[0112] RLS particles provide a tool for analyzing and understandingsmall fluid volume deposition and dynamics on glass slides during arraygeneration. Individual RLS particles can be seen using simpleinstrumentation to obtain an appropriate level of magnification. For pintransfer, where there is generally direct contact between the pin thatcarries the fluid held by surface tension and the slide surface upondeposition, distribution of fluid and the probe contained therein israrely uniform. Furthermore, the distribution of probe frequentlychanges with drying as local sub-regions within the deposited fluidrapidly form. This effect causes non-uniform distribution of probeacross the spot area. As such, the behavior of the probe acrossdifferent areas of the spot is inconsistent during hybridization. Thiseffect also can cause spot instability or “flaking” as areas of the spotthat dry last generally have high levels of salt and probe, creatingareas of differential binding on the slide. Different glass surfacemodifications and/or drying conditions either partially reduce orexacerbate this effect. The effects of specific conditions or changescan be determined using RLS particles, which may, for example, beattached to probe before or after deposition.

[0113] Other spotting methods are somewhat improved with respect toprobe distribution since no contact pin is used to immediately establishliquid distribution differences upon deposition. Nevertheless, currentmethods do not allow one to examine, with a high degree of resolution,the parameters associated with spotting and drying and probedistribution that may still occur to some level with syringe-solenoidand piezoelectric deposition. Apart from this, there are other issuesrelated to volume uniformity and port clogging in these two approachesin different applications. In this case, RLS particles are useful indeveloping formulations for spotting that affect uniform drying andimproved drying dynamics, in conjunction with determination of theappropriate substrate surface properties to establish optimal assayperformance. Typically, glass, silicon, or plastic substrates are usedwith a variety of surface chemistries including but not limited toamino, aldehyde, carboxylic acid, thiol, and functionalized silanes. RLSparticles can also be used to determine the distribution of “functional”or hybridizing probe to its specific target under defined conditions.This aspect provides a method for not only determining the physicaldistribution of probe (nucleic acid, protein or any molecular componentof a binding pair or complex) within the deposited area, but also theoperational performance of the probes across the array feature.

[0114] RLS particles are also valuable tools for design, development andimplementation of an arraying device. This is particularly the case withrespect to pin, syringe or piezoelectric jet head design and qualitycontrol of pin manufacturing and performance. The ability to observesmall fluid volumes and fluid dynamics using RLS particles is alsoimportant for developing robotic and fluid handling systems forreproducibly generating microarrays.

[0115] As indicated throughout the present description, the presentmethods are useful in many different applications. In the specificexample of array production, exemplary system and process parametersthat can be evaluated using RLS particle detection include, for example,spotting buffer formulation, pin selection, probe concentrations,relative humidity, post-spotting treatment, probe/feature stability tovarious experimental steps, and the like. These and other parameters canall be monitored in functional system development assays with a highlevel of resolution.

[0116] In connection with the use of light scattering particles forimproving array drying parameters, an example is provided by thedeposition of gold particle spots (e.g., 3 mm diameter) on glass slidescoated with a gold particle binding agent. Careful observations weremade of the manner in which water evaporates from the spot and theeffect that this evaporation has on the final spatial distribution ofparticles in the spot. Initially, evaporation occurs at the periphery ofthe spot, causing a current of particles to the periphery resulting inthe deposition of particles in an intense ring at the periphery. Theserings are often seen in microarrays from different companies, indicatingthat this evaporation pattern also occurs when DNA probes are depositedin microspots. As evaporation proceeds, the particle current to theperiphery ceases and a new current causes the particles to concentratein a halo that is positioned between the periphery and the center of thespot. As total evaporation is approached, the halo concentrates on aspot which results in an intense area of particles in the final spot.This type of nonuniform pattern was also detected in commercial DNAchips that were examined, indicating that the deposition of DNA probesby evaporation results in DNA probe patters similar to the ones observedwith RLS particle depostion.

[0117] Evaporation can be prevented by use of a humid chamber. In thiscase, the deposition of particles is diffusion dependent. Sincediffusion is very slow, it takes overnight incubation and this stilldoes not guarantee uniform spots. Thus, evaporation seems to be the bestmethod for quickly bringing down the particles to a the glass surface.In order to improve the drying process to provide better uniformityacross individual spots, a series of process modifications wereperformed and the effects tested using light scattering particles.

[0118] Studies on the evaporation process led to the idea that uniformspots might be obtained with use a two solvent system, one whichevaporates rapidly (e.g., water) and the other (e.g., DMSO) whichevaporates more slowly. Rapid evaporation of one of the solvents resultsin particles in a very thin film of the second solvent. In this thinfilm, particles can reach the surface by diffusion in a short time.While the use of a two solvent system improves uniformity, thedistribution of gold particles in a spot is not always completelyuniform.

[0119] In further experiments, a polymer was added gelatin to thetwo-solvent system as an agent for binding gold particles to the glasssurface; with this addition, the spots were very uniform. It appearsthat as the first solvent evaporates, the polymere is concentrated intoa viscous gel in the second solvent and that this inhibits particlecurrents. The particles thus maintain a uniform distribution in thesecond solvent and deposit on the surface in a uniform pattern. In anexample using 80 nm gold particles with Ficoll, spots with uniform goldparticle spatial distributions were obtained, but the particles had anorange light scattering color instead of the usual green lightscattering color of 80 nm gold particles in air. However, after removingthe Ficoll by washing, the spots displayed the expected green lightscattering color.

[0120] Closer examination of the process revealed still furtherimprovements. The washing process was examined in more detail by addinga small volume of water to the spot while viewing the spot in a darkfield microscope. Observation revealed that many of the particles cameoff during washing. It appears that if the Ficoll concentration is high,many of the particles are trapped within the Ficoll gel matrix and notactually attached to the glass surface. However, within a certain lowFicoll concentration range, the spots are uniform and very few particlescome off the spot during the washing step.

[0121] The process just described illustrates the application of thepresent methods for selecting materials, optimizing concentrations ofmaterials, and improving process steps, resulting in improved spotuniformity.

[0122] A further illustration is directed to buffer selection, e.g., forselecting buffers for a washing step in an assay. In immuno and DNAprobe assays on solid surfaces, the ability to see individual particleswas very helpful in developing buffers with which to remove backgroundnon-specifically bound particles without affecting the specificallybound particles. On initial exposure of the solid phase to particlesolution, typically a large number of particles become non-specificallybound to the solid phase material and/or to material bound on a samplespot. Indeed, in some cases, the surface concentration ofnon-specifically bound particles can exceed that of specifically boundparticles, apparently due to electrostatic effects. In order to select abuffer solution, the relative effects of candidate buffers on thenon-specifically bound particles and specifically bound particles can becompared. While viewing an RLS labeled spot, buffer is added. By lookingat individual particles, the relative effects of the buffer on particleson the spots versus particles in the surrounding background areas can bedetermined. As indicated, those effects can then be compared fordifferent buffers. This approach can similarly be applied to differenttypes of solutions and solution components, e.g., relating to binding,reaction, washing, and/or detection steps.

[0123] A particular example of the utility of these methods relates todetermination of the surface distribution of functional immobilizedoligonucleotide capture probes on a solid phase surface. In thisapplication, RLS particles directly or indirectly attached tocomplementary molecules are used to locate and gauge the distribution offunctional (able to efficiently hybridize) probes after spotting andexperimental processing. One can easily see and quantify thedistribution and uniformity of functional probes resulting from aparticular experimental protocol to minimize deleterious effects ofsmall-volume drying, local salt gradients, crystallization, and thelike.

[0124] In connection with production and use of arrays (and other deviceor assay formats that involve some form of binding of molecules (e.g.,biomolecules such as nucleic acids and polypeptides) to solid phasesurfaces, methods for labeling molecules such as nucleic acids and/orother biomolecules for analysis are numerous, but generally fall intotwo broad categories known as direct and indirect labeling.

[0125] For direct labeling, RLS particles can replace fluorescent tagsand/or radioisotopes, and be incorporated directly into nucleic acidsand/or polypeptides and/or other biomolecules. In this case, theparticles are directly attached to the molecule of interest, e.g.,attached to a nucleoside triphosphate or analog thereof and incorporatedin an oligonucleotide. The RLS particle labeled nucleic acids and/orother molecules can be distributed onto a solid surface, such asmicroarray chips. The number and distribution of the RLS particles is anindicator of the number, distribution, and homogeneity of the depositednucleic acids and/or other biomolecules. Such distribution andhomogeneity can then be detected using the RLS particles as described,preferably using simple instrument, or even by eye with appropriatemagnification.

[0126] For indirect labeling, the RLS particle labeled molecule, e.g.,nucleic acids and/or other biomolecules, can also be used as probes fordetecting a molecule or molecules of interest. In general, such probesutilize specific binding pair interactions. Common examples are inhybridization or binding assays. In these cases, the determination ofRLS signals indicates the distribution of functional biomoleculesselectively interacting with the probes. RLS particles can be associatedwith probe molecules in various ways (also applicable to associating RLSparticles with molecules of interest). Typically the RLS particles areeither indirectly associated with probe biomolecules such as nucleicacids or antibody probes through hapten binding effected by haptenincorporated into the probes, or by directly coupling the probes to theRLS particles. Hapten is incorporated into probe by either enzymatic orchemical labeling, and subsequently RLS particles coated withstreptavidin or antibodies or other molecules that specifically bind tothe introduced hapten are attached to the probe, Alternatively, asindicated, specific probes can be directly coupled to RLS particles byattaching the nucleic acid, antibody, or other biomolecule probes to thesurface of RLS particles. In addition, RLS particles can be directlyintroduced into nucleic acid probes by incorporation labeling in thepresence of the appropriate ribo- or deoxyribonucleosidetriphosphate-derivatized RLS particles.

[0127] Dispensing and Mixing of Micro Volumes

[0128] RLS particles can also play an important role in development ofinstruments and optimization of methods for micro-dispensing fluids.This use may become especially important as combinatorial chemicalsynthesis moves toward miniaturization in concert with emerging lowvolume high throughput screening approaches (HTS) (e.g., 1564, 3456 and9600 well formats). With respect to HTS, RLS particles are useful tooptimize procedures associated with micro-volume reagent introductionand mixing.

[0129] The demonstrated ability to visualize individual RLS particlesunder high magnification using RLS microscope detection instrumentestablishes the usefulness of the present methods in small volume deviceand process development, testing, and quality control. One can easilydistinguish by eye individual particles both in solution undergoingBrownian motion and as single particles immobilized to solid phase. Thissimple observation can be done, for example, by taking uncoated RLSparticles bearing a net negative surface charge and spotting dilutesolutions onto poly-lysine coated microscope slides. This same type ofexperiment has been done in solution using different sizes of RLS goldparticles. In this case, one cannot only see individual particlesdistinctly, but the different colors and intensities can also beobserved.

[0130] In a particular application, numerous experiments have beenconducted using RLS particles to optimize spotting of nucleic acids formaking microarrays. Throughout these experiments, uniformity ofdistribution of probes on a microarray feature and resulting signal wasan emphasis. Indeed the ability to observe the signal on features infine detail has also been conducted using many different systems usingRLS particle detection. Most microarray spots developed without thisimportant tool and process development advantage are more likely to beof significantly poorer quality, because of the limited ability tomonitor and develop optimal equipment and processes. This has been notedin evaluation of cDNA and PCR product arrays spotted on glass slides aswell as for many other array systems. RLS particles have also been usedto examine the effect of different drying conditions on capture probedistribution and uniformity.

[0131] With regard to microfluidics, similar observations regardingindividual particle detection have been made and applied. In one type ofexperimental demonstration, RLS particles were run through amicro-channel device by passive capillary action and detected using theRLS microscope instrument. Under these conditions, one can clearly seeindividual particles suspended in flow. Further, one can observedifferential flow rates of individual particles as a function of channelposition with respect to the channel wall. At cross-channel junctions,one can also readily observe RLS particles in flow into the crosschannel at a reduced rate relative to the main component flow direction.One can quantitate component flow rates using real time analysis withconventional or high speed video camera linked to a personal computerand confinement analyzers and software.

[0132] Surface/Matrix Characterization

[0133] RLS particles can also be used to examine and/or quantitatevarious surface characteristics of solid surface substrates, and matrixproperties of membranes and other porous substrates. Suchcharacteristics and properties include but are not limited to physical,chemical and/or electrical properties such as flatness, texture,porosity, surface charge, surface charge uniformity, hydrophobicity,hydrophilicity, and chemical reactivity in addition to other properties.One of ordinary skill in the art will appreciate the utility of a small,stable, highly intense, particulate label, the surface of which can besystematically configured to provide multiple distinct properties, tointerrogate and quantify label interaction with the substrate surface ormatrix.

[0134] For example, the charge properties of a substrate surface can beexamined by treating the surface with RLS particles having a distinctnet surface positive or negative charge under conditions of low ionicstrength. Such RLS particles may be prepared by coating the surface ofthe RLS particles with different mixtures of charged and/or nonchargedpolymers such at poly-lysine (positively charged polymer), polyethyleneglycol (charge-neutral polymer) and/or a polymer of acrylic acid(negatively charged polymer). After treatment of the substrate surfacewith surface-charge-modified RLS particles under conditions that affectparticle-substrate binding through charge-charge interaction, one canquantitate the uniformity of the charged substrate surface byquantitating the number and distribution of RLS particles on thesurface. This would most typically be done at high resolution under darkfield illumination conditions using a microscope instrument fitted witha CCD camera coupled to image and statistical analysis software tofacilitate quantification of RLS particles on the substrate surface.

[0135] As another example, uniformity of distribution of reactive groupson a surface or in a porous substrate (e.g., a functionalized surface)can be determined by selecting a chemically appropriate molecule andreacting it with the reactive groups. The chemically appropriatemolecule is additionally selected to provide binding to a moiety linkedor linkable to RLS particles. As an example, the molecule reacted withthe surface can provide a binding site for an antibody that is thenrecognized by a second antibody attached to an RLS particle. Numerousother binding arrangements can also be used. The number and/ordistribution of bound RLS particles is then determined, and used as anindication of the number and/or distribution of functional groups on thesurface or matrix.

[0136] Such approaches are particularly useful for analysis and qualitycontrol of various glass or plastic substrates which may have beenmodified using functionalized silanes, polymers, three dimensionalmatrices or porous materials to enhance binding of molecules to thesubstrate surface. Similar approaches can be applied to examineproperties of membrane or other porous substrates related to function orperformance in various applications including binding of molecules. Onecan also use RLS particles to examine the changes in substrate surfaceor porous matrix properties as a function of processing the substrate ormatrix through one or more steps in an experimental procedure. Forexample, the effect on various properties with treatment of a solidglass or plastic substrate with heat, high salt buffers, organicsolvents, blocking agents, detergents, in addition to other treatments,can be monitored using RLS particles.

EXAMPLES Example 1 Preparation of a 16 nm Gold Particle Suspension

[0137] 2.5 ml of sterile water was added to 0.1 g HAuCl₄.3H₂O to form a4% HAuCl₄.3H₂O solution. The solution was centrifuged to removeparticulate matter. In a separate flask, 10 ml of sterile water wasadded to 0.1 g. of sodium citrate to form a 1% sodium citrate solution.The citrate solution was filtered through a 0.4 μm polycarbonatemembrane filter to remove particulate matter. To a very clean 250 mlErlenmeyer flask, 100 ml of sterile water and 0.25 ml of the 4%HAuCl₄.3H₂O was added. The flask was placed on a stir hot plate at asetting of 4 and covered with a 100 ml beaker. When the mixture startedboiling, 2 ml of the 1% sodium citrate was added. The mixture solutionwas boiled for 30 more minutes and then cooled to room temperature andsterile water was added to bring the total volume to 100 ml. The finalgold concentration is about 0.005% and particle concentration is1.2×10¹² particles/ml, assuming that all the gold salt was converted togold particles.

Example 2 Stabilization of Metal Particles with Polvethylene

[0138] 1 gram of PEG (MW 20,000) was added to 100 ml of sterile water toform a 1% PEG solution and the solution was filtered through a 0.4 μmpolycarbonate filter using a 50 ml syringe. To stabilize a given volumeof particles, the volume of particle solution was added to a volume of1% PEG solution that gives a final PEG concentration of 0.1%.

Example 3 Preparation of 30 nm Silver Coated Particles from 5 nmDiameter Gold Particles

[0139] 10 ml of sterile water was brought to a boil in a 30 ml beaker. 2mg of gelatin was then added slowly and the solution was allowed tocontinue to boil with stirring until all of the gelatin was dissolved.The solution was then cooled to room temperature. 2 ml of a 47% citratebuffer pH 5 was added. 0.18 ml of a solution containing 5 nm goldparticles (at a concentration of about 0.005% gold, 3.8×10¹³ goldparticles/ml) was added followed by the addition of 3 ml of a 5.7%hydroquinone solution. The mixture was mixed well, followed by additionof sterile water for a final volume of 20 ml. 50 μl of a 4% silverlactate solution was added in 10 μl increments and the mixture wasstirred rapidly by hand. The final silver concentration is about 0.005%and the final silver coated particle concentration was about 3.4×10¹¹particles/ml. Assuming that all of the added silver had depositedequally on each gold particle, the particle size was calculated to be 30nm. After the final addition, the sol appeared bright yellow in roomlights. In bulk solution, the light scattered by a diluted volume of thesol contained in a 6×50 mm glass tube was blue when illuminated by anarrow beam of white light. When a dilution of the silver sol wasexamined microscopically with an RLS microscope instrument through a 10×objective and 12.5 eyepiece, a mixture of bright particles withdifferent colors could easily be seen. The particles dominant in numberwere purple-blue particles. Yellow, green and red particles were alsopresent. By adjusting the concentration of the 5 nm diameter goldparticles used in the procedure described here, many sizes of silvercoated particles can be made, e.g., with diameters in the range 20 to100 nm.

Example 4 Preparation of Larger Diameter Gold Particles from 16 nmDiameter Particles

[0140] A 2.4% solution of hydroxylamine hydrochloride was made by adding24 mg of hydroxylamine hydrochloride to 1 ml of sterile water, mixingand then filtering through a 4 μm polycarbonate membrane filter attachedto a 10 ml syringe. A solution of 4% HAuCl₄.3H₂O was made by adding 2.5ml of sterile water to 0.1 g HAuCl₄.3H₂O in a test tube mixing and thencentrifuging to remove particulate matter. 25 ml of sterile water wasadded to a 250 ml Erlenmeyer flask, followed by addition of the volumeof 16 run gold particles shown in Table 1 depending on the desiredparticle size. Next the volume of the 4% HAuCl₄.3H₂O solution specifiedin Table 1 was added. Finally sterile water was added to bring the totalvolume to 100 ml. Then the volume of the hydroxylamine hydrochloridesolution specified in Table 1 was added with rapid hand stirring and themixture was allowed to sit for 30 minutes. Within seconds after addingthe hydroxylamine hydrochloride solution, the solution turned from aclear, slightly pink color to a final clear red or murky brown color,depending on particle size. The smaller sizes give red coloredsolutions. TABLE 1 Desired 16 nm Hydroxyl- Au Particle Gold HauCL₄.3H₂Oamine Diameter, nm Sol, ml Solution, ml Solution ml 40 6.4 0.234 1.25 601.9 0.245 1.25 80 0.8 0.248 1.25 100 0.41 0.249 1.25

[0141] Larger diameter particles were prepared following the sameprocedure as described above, but using the specified volumes ofsolutions as described in Table 2 and using the 100 nm diameter gold(Au) particle solution instead of the 16 nm gold solution. TABLE 2Desired Au 16 nm 4% Hydroxylamine Particle Gold HauCl₄.3H₂O SolutionDiameter, nm Sol, ml Solution, ml ml 200 12.5 0.219 1.25 400 1.56 0.2461.25 600 0.436 0.249 1.25 800 0.195 0.25 1.25 1000 0.1 0.25 1.25 20000.012 0.25 1.25

Example 5 Preparation of a Silver Coated Particle from 16 nm GoldParticles

[0142] 25 ml of sterile water was added to a 250 ml Erlenmeyer flaskfollowed by the addition of 6.4 ml of a 0.005% 16 nm gold particle soland the resulting solution was mixed. 0.234 ml of a 40 mg/ml L(+) LacticAcid silver salt solution was then added. A deep purple color wasimmediately seen. Enough sterile water was then added to bring the totalvolume to 100 ml. While rapidly stirring by hand, 1.25 ml of a 24 mg/mlsolution of Hydroxylamine Hydrochloride was added and the resulting solappeared lavender silver in color. A small drop of the sol was placed ona glass slide, covered with a cover glass and examined with an RLSmicroscope instrument. Red, green, purple, and yellow particles wereseen. The scattered light color of a dilute solution of these particlesin a test tube with white light illumination was ice blue.

Example 6 Field Flow Fractionation Design and Fabrication

[0143] Field Flow Fractionation (FFF) systems can be made in both Macroor Micro formats. Macro systems typically have channels 30-60 cm inlength, a height of around 130 μm, and a width of 2 cm. (B. K. Gale, K.D. Caldwell, A. B. Fraiser, “A Micromachined Electrical Field-FlowFractionation (μEFFF) System, IEEE Transactions on BiomedicalEngineering, Vol 45, No 12, pp. 1459-1468, December 1998). Macro systemsare large enough to be manufactured through traditional processes suchas plastic injection molding, and film lamination of clear plastics suchas polystyrene, polycarbonate, acrylic and mylar.

[0144] Micro devices manufactured through micromachining are typically 6cm in length, 8 mm in width and 10-30 μm in height. Both Macro and Microsystems generally utilize width to height ratios of channel dimensionsof greater than 100:1 to minimize edge effects, approximating flowbetween two infinite parallel plates. Micro devices are typicallyfabricated with silicon or glass substrates, and can be patterned withvacuum deposited electrodes for manipulation and detection of the fluidwithin the channel electrically. These electrodes can be pure metalssuch as gold, tin or platinum, or alloys such as indium-tin oxide. MicroFFF devices are manufactured with semiconductor process such asphotolithography using “spin on” polyamide coatings over the substrate,such as SU8 epoxy to create the space for the channels. Individualcomponents or the entire channel may be coated with a wetting agent orsurfactant to change the fluid dynamics of the channel. Coatings mayalso be used to provide biocompatibility between the device and sample.Those skilled in the art are familiar with various coatings and methodsof coating.

[0145] There are a number of different types of FFF systems based on thetype of forces or fields applied across the channel from top to bottom.This force results in the fractionation of particles of different sizeor charge density. These forces can be applied by temperature gradients,sedimentation (both gravitational and centrifugal), cross-flow,magnetic, and particle charge density (electrical). Fluid flow alonglength is typically accomplished using a small peristaltic or syringepump with a flow rate controller, however, capillary flow systems havealso been developed. Flow rates for FFF systems are determined by thedesired resolution, channel dimensions and the force being applied.Normal operating flow rates are typically lcm/sec and lower.

[0146] Light scattering particles can be utilized in the design andmanufacture of FFF systems, for example, to analyze fluid flow withinthe device, separation patterns, manufacturing quality control, and flowinto and out of the device.

Example 7 Detection of RLS Particles in a Microchannel

[0147] This example demonstrates the detection of light scatteringparticles in a microchannel device, but is demonstrative of similardetection in a variety of different types of devices.

[0148] 0.05 μL of 80 nm anti-biotin coated gold RLS particles (1:10mixture of 5OD particles:DI water), were injected into a flat, glassmicrochannel having clear top and bottom surfaces, a cross-sectionalarea of 1.25×10⁻³ cm² (25 μm×5.0 mm), length of 500 mm, and a totalvolume equal to 7.5×10⁻³ mL. The carrier fluid was deionized water witha flow rate of 0.6 mL/hr (0.13 cm/s).

[0149] Light was provided by a trans-illumination system using a focusedpolychromatic light source, directed to the channel through a prismoptically coupled to the bottom of the channel arranged to provide darkfield illumination, Particles in suspension were introduced into themicrochannel, and flowed in the microchannel. Flow can be generated in avariety of ways, including a variety of force and capillary flowapproaches. While this example utilized trans-illumination,epi-illumination has also been demonstrated in microchannel applications(and is applicable to a variety of other applications). Suchepi-illumination (or other illumination technique with illumination anddetection from the same side of the surface or device) can be used withtransparent channels, or with channels (likewise for other types ofmicrodevices) with only one transparent surface.

[0150] Images were acquired using a microscope objective-based viewingsystem fitted with a CCD video camera. Four time-lapse images are shownin FIG. 3, with the first image located at the top. The interval betweenimages was 86 msec. The axis of the channel is oriented vertically inthe images.

[0151] This example demonstrates that light scattering particles can bereadily introduced and detected in microdevices. One of ordinary skillin the art will recognize that the detection can be readily adapted to alarge variety of other microdevices of varying scale, as well as to manymacrodevice applications.

[0152] All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

[0153] One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themethods, variances, and compositions described herein as presentlyrepresentative of preferred embodiments are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art, which areencompassed within the spirit of the invention, are defined by the scopeof the claims.

[0154] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention. For example, using other light scattering particles, and/ormicro devices are all within the scope of the present invention. Thus,such additional embodiments are within the scope of the presentinvention and the following claims.

[0155] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms for other embodiments. The terms andexpressions which have been employed are used as terms of descriptionand not of limitation, and there is no intention that in the use of suchterms and expressions of excluding any equivalents of the features shownand described or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

[0156] In addition, where features or aspects of the invention aredescribed in terms of Markush groups or other grouping of alternatives,those skilled in the art will recognize that the invention is alsothereby described in terms of any individual member or subgroup ofmembers of the Markush group or other group.

[0157] Where a component or limitation is described with a variety ofdifferent possible numbers or dimensions associated with that componentor limitation, in additional embodiments, the component or limitation isin a range specified by taking any two of the particular values providedas the endpoints of the range. The range includes the endpoints unlessclearly indicated to the contrary.

[0158] Thus, additional embodiments are within the scope of theinvention and within the following claims.

What is claimed is:
 1. A method for determination of a dynamic propertyof a fluid volume, comprising, determining the distribution or locationor both of at least one light scattering particle in said fluid volumeby detecting light scattered from said at least one particle.
 2. Themethod of claim 1, wherein said dynamic property is flow rate.
 3. Themethod of claim 1, wherein said dynamic property is particledistribution in said fluid volume.
 4. The method of claim 3, whereinprobes are present in said fluid volume and said particle distributionis indicative of the distribution of said probes in said fluid volume.5. The method of claim 4, wherein said distribution of probes is on asolid phase surface.
 6. The method of claim 1, wherein said dynamicproperty is uniformity of drying on a solid surface.
 7. The method ofclaim 1, wherein said dynamic property is a flow pattern in a device orportion of a device, said device being an article of manufactureincluding one or more channels or reservoirs for fluid.
 8. The method ofclaim 7, wherein said dynamic property is fluid mixing being evaluatedin one or more portions of said device or through the entire device,said portions being selected from the group consisting of a mixingchamber, a port, a flow channel, a pump, a valve, and a flow channelintersection.
 9. The method of claim 1, wherein said fluid volume is ina small volume device.
 10. The method of claim 9, wherein said smallvolume device is selected from the group consisting of a micro volumedevice, a nano volume device, and a pico volume device.
 11. The methodof claim 9, wherein said small volume device is selected from the groupconsisting of an array chip, array plate, or array slide;
 12. The methodof claim 9, wherein said small volume device is a membrane or porousmatrix.
 13. The method of claim 9, wherein said small volume device isselected from the group consisting of a pump; a port, a channeljunction, and a valve.
 14. The method of claim 9, wherein said smallvolume device comprises an array comprising a plurality of features andhas deposited on each feature a volume of 10 pL to 10 nL.
 15. The methodof claim 9, wherein said small volume device comprises an arraycomprising a plurality of features and has deposited on each feature avolume of 10 nL200 nL.
 16. The method of claim 9, wherein said smallvolume device comprises an array comprising a plurality of features andhas deposited on each feature a volume of 200 nL to 2 microliters. 16.The method of claim 9, wherein said small volume device is amicrochannel device, comprising at least one microchannel of sufficientsize to allow fluid flow.
 17. The method of claim 1, wherein said atleast one particle comprises a plurality of distinguishable particles.18. The method of claim 17, wherein said plurality of distinguishableparticles is used to analyze mixing of fluids from two differentsources.
 19. A method for analyzing deposition characteristics offeatures on an array, comprising depositing at least one fluid volume ona portion of a solid substrate, wherein said fluid volume contains aplurality of light scattering particles; detecting the distribution ornumber or both of said light scattering particles by detecting lightscattered from said particles, wherein the distribution or number orboth of said particles is indicative of one or more depositioncharacteristics.
 20. The method of claim 19, wherein said depositioncharacteristic is uniformity of deposition, wherein said uniformity isevaluated by determining at least one of the properties selected fromthe group consisting of a 2-dimensional distribution of particles withinat least one feature, deposition volume, and uniformity of particlenumber in deposited fluid volumes.
 21. The method of claim 19, whereinsaid deposition characteristic is a drying pattern.
 22. The method ofclaim 19, wherein said array has bound thereto a plurality of probemolecules and said distribution of particles is indicative of thedistribution of probe molecules deposited on said array.
 23. The methodof claim 22, wherein said distribution of probe molecules is adistribution during drying of said at least one feature.
 24. The methodof claim 22, wherein said distribution of probe molecules is adistribution during or after post-spotting processing of said at leastone feature.
 25. The method of claim 19 wherein said depositioncharacteristic is indicative of functional binding on said at least onefeature, wherein said functional binding is a nucleic acid-probehybridization, protein-protein interaction, or ligand-receptor binding.26. The method of claim 19, wherein said array comprises at least 10features.
 27. The method of claim 19, wherein said array comprises atleast 100 features.
 28. The method of claim 19, wherein said arraycomprises at least 1000 features.
 29. The method of claim 19, whereinsaid array comprises at least 10,000 features.
 30. The method of claim19, wherein said array comprises greater than 10,000 features.
 31. Amethod for analyzing fluid flow in at least one portion of a smallvolume device, comprising illuminating a suspension of light scatteringparticles in at least one portion of said device; and detecting thepresence of said light scattering particles as an indication of saidfluid flow.
 32. The method of claim 31, wherein a plurality of differentlight scattering particles are inserted in said device, and saidplurality of different particles are detected as an indication of saidfluid flow.
 33. The method of claim 31, wherein said at least oneportion is a plurality of portions of said device.
 34. The method ofclaim 31, wherein said flow is detected using extended exposure, wherebysaid light scattering particles provide flow tracers.
 35. A method foranalyzing at least one characteristic of a solid or porous substrate,comprising treating at least a portion of a sample of said substratewith at least one fluid volume containing a plurality of lightscattering particles; and detecting the distribution or number or bothof said light scattering particles on said at least a portion of saidsample by detecting light scattered from said particles, wherein thedistribution or number or both of said particles is indicative of saidat least one characteristic.
 36. The method of claim 35, whereinsubstrate is a solid substrate and said characteristic is a surfacecharacteristic.
 37. The method of claim 35, wherein said substrate is aporous matrix.
 38. The method of claim 36, wherein said at least onecharacteristic is selected from the group consisting of surfaceuniformity, uniformity of one or more surface coatings, uniformity ofsurface charge, uniformity of surface hydrophilicity, uniformity ofsurface hydrophobicity, and uniformity of surface charge density. 39.The method of claim 35, wherein said substrate is selected from thegroup consisting of a glass substrate, a functionalized glass substrate,a plastic substrate, a silicon substrate, a membrane substrate, and ametallic substrate.
 40. The method of claim 37, wherein said porousmatrix is selected from nitrocellulose, polyvinylidene fluoride, andnylon.
 41. The method of claim 37, wherein said at least onecharacteristic is selected from the group consisting of matrixuniformity, uniformity of one or more coatings, uniformity of charge,uniformity of hydrophilicity, uniformity of hydrophobicity, anduniformity of charge density.